Selenium is an essential trace element, which has provoked considerable interest due to the recent identification of prokaryotic and eukaryotic proteins that contain the amino acid, selenocysteine. Incorporation of selenocysteine into these proteins requires a novel translation step in which UGA specifies selenocysteine insertion instead of termination. This is conferred by a complex interaction between specific components of the translation machinery and secondary structures in selenoprotein mRNAs, termed SECIS elements. The specific aims of this proposal are 1) to investigate differential SECIS element function for different regions of the mRNA for selenoprotein P (see below), and to investigate the factors contributing to the differential activities of these elements and to the "hierarchy of selenoprotein synthesis", 2) to investigate the effects of coding region sequences on selenocysteine incorporation efficiency, 3) to investigate the effects of translation initiation rates on selenoprotein synthesis, and 4) to determine the optimal levels and ratios of the factors involved in this process, with the goal of developing a system for more efficient selenoprotein synthesis. The identification of selenocysteine in numerous mammalian and lower eukaryotic proteins during the past decade has provided new insights into the functions of this trace nutrient. Studies of selenocysteine incorporation in type 1 iodothyronine deiodinase opened the door for investigation of the requirements for eukaryotic selenoprotein synthesis, and the features that distinguish this pathway from the mechanism in prokaryotes. More recently, studies of the biosynthesis of selenoprotein P, which contains 10 - 17 selenocysteine residues, have provided an ideal system in which to investigate the efficiency of selenocysteine incorporation. While significant progress has been made in recent years, investigation of the complex interplay between the mRNA secondary structures, a novel SECIS binding protein, the selenocysteine-specific elongation factor, and other components of the translational machinery remains foremost among the tasks ahead. Examination of physiological circumstances contributing to tissue-specific differences in selenocysteine incorporation will help to explain how the animal has adapted to maximize utilization of this trace element, with crucial implications for advancing our understanding of the roles of selenium in health and disease. Finally, studies of eukaryotic selenoprotein synthesis have provided unexpected insights into the mechanisms of translation and termination of protein synthesis. Continuing studies of this process will surely unveil new and exciting information about the complex workings of the translation machinery.